A polypeptide inhibitor of calcineurin blocks the calcineurin-NFAT signalling pathway in vivo and in vitro

Abstract

Calcineurin (CN) controls the immune response by regulating nuclear factor of activated T cells (NFAT). Inhibition of CN function is an effective treatment for immune diseases. The PVIVIT peptide is an artificial peptide based on the NFAT-PxIxIT motif, which exhibits stronger binding to CN. A bioactive peptide (named pep4) that inhibits the CN/NFAT interaction was designed. Pep4 contains a segment of A238L as the linker and the LxVP motif and PVIVIT motif as CN binding sites. Pep4 has strong binding capacity to CN and inhibits CN activity competitively. 11-arginine-modified pep4 (11 R-pep4) inhibits the nuclear translocation of NFAT and reduces the expression of IL-2. 11 R-pep4 improves the pathological characteristics of asthmatic mice to a certain extent. The above results indicated that pep4 is a high-affinity CN inhibitor. These findings will contribute to the discovery of new CN inhibitors and promising immunosuppressive drugs.

Keywords: Calcineurin; NFAT; enzyme inhibitor; polypeptide.

Figure 1.

Pep4 strongly binds CN. (A) Pep4 binds CNA from mouse brain lysates in GST pull-down assays. GST-pep4, GST-pep1s, and GST-pep2 were incubated with CN from the mouse brain, CN monoclonal antibody was used to observe the combined CN by western blotting (upper panel), and the same amount of mouse brain lysates was added to each group middle panel). Western blotting was performed on the fusion protein with GST antibody to confirm the amount of GST-fusion protein added in each group (bottom panel). (B) The optical density of CN bound to pep4, pep1s, and pep2 was measured. The histogram reflects the relative ability of combining CN. Data are presented as the mean ± SEM of three independent experiments. **p < 0.01 and ***p < 0.001 compared with the pep4 group. (C) Schematic diagram of pep4 and its mutants. Pep4 is composed of pep1s, linker and pep2. The important binding sites of pep1s and pep2 were mutated to obtain mutants 1, 2 and 3. (D) Pep4 and its mutants bind CNA from mouse brain lysates in GST pull-down assays. The pull-down experiment was performed under the same experimental conditions as B. (E) The optical density of the CN bound to pep4 and its mutants was determined. The histogram reflects the relative ability of combining CN. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the pep4 group. (F) The binding of pep4 to endogenous CN in HEK293 cells. GFP empty plasmids or GFP-pep4 plasmids were transfected into HEK293 cells for 24 hours. The cell lysate was subjected to immunoprecipitation experiments with GFP antibody. The final lysate was subjected to western blotting detection, and CN monoclonal antibody was used to detect the binding of CN in each group. The IgG group and GFP group serve as negative controls. (G) A238L, pep3 and pep4 bind CNA from mouse brain lysates in GST pull-down assays. The pull-down experiment was performed under the same experimental conditions as B and D. (H) The optical density of the CN bound to A238L, pep3 and pep4 was determined. The histogram reflects the relative ability of combining CN. Data are presented as the mean ± SEM of three independent experiments.

Figure 2.

Pep4 competitively inhibits the enzymatic activity of CN. (A) The effect of pep4 on CN enzyme activity in the mouse brain. The horizontal axis represents the concentration of pep4, and the vertical axis represents the inhibition degree of enzyme activity. (B) The effect of pep4 and its single binding motif on CN enzyme activity. Pep1s, pep2 or pep4 at the same concentration of 25 nM were incubated to detect the enzyme activity of CN. The horizontal axis represents the different short peptides, and the vertical axis represents the enzyme activity of CN. (C) Kinetic analysis of CN in brain extracts with or without pep4. Lineweaver-Burk plots of activity against substrate concentration in mouse brain extracts.

Figure 3.

Cell-permeable pep4 can block the ionomycin-induced nuclear translocation of NFAT1 and the gene expression of the CN/NFAT downstream pathway. (A) Immunofluorescence staining of Jurkat cells incubated with FITC-11R-pep4. Green represents FITC-11R-pep4. The nucleus is stained with Hoechst 33258. Scale bar, 10 μm. (B) The curve between incubation time and fluorescence intensity of FITC-11R-pep4. Incubation time is 1–14 h. (C) Average fluorescence intensity of FITC-11R-pep4 at different incubation times determined by flow cytometry. The incubation times were 1, 3, 5 and 7 h. (D) Average fluorescence intensity of FITC-11R-pep4 at different concentrations after 6 h incubation by flow cytometry. The concentrations of FITC-11R-pep4 were 1, 2, 5, and 10 μM. (E) 11R-pep4 inhibits NFAT1 from entering the nucleus. HEK293 cells are incubated with 11R-pep4. The nuclei and cytoplasm of HEK293 cells were separated and analysed by western blotting. NFAT1 monoclonal antibody was used to detect the amount of NFAT1 in the nucleus and cytoplasm, and β-actin and lamin B1 were used as the loading controls. CsA served as the positive control. (F) The optical density of NFAT1 in the cytoplasm and nucleus was measured. The histogram reflects the relative content of NFAT1 in each group. Data are presented as the mean ± SEM of three independent experiments; ***p < 0.001 compared with the ionomycin group. (G) 11R-pep4 inhibits IL-2 mRNA expression. Jurkat cells were pre-treated with different concentrations of 11R-pep4 and stimulated with 1 µM ionomycin and 50 mg/mL PMA. Total RNA was extracted for quantitative real-time PCR to detect the mRNA expression of the IL-2 gene, and CsA served as the positive control.

Figure 4.

11 R-pep4 can alleviate the inflammatory response in asthmatic mice. (A) Schematic diagram of the mouse asthma experimental design. Male BALB/c mice were selected and sensitised by intraperitoneal injection of 10 μg OVA on the 7th and 14th days. Mice were challenged with 1% OVA for four consecutive days from the 21st day to construct an acute asthma model. Then, 11R-pep4 was administered before each OVA challenge. The mice were sacrificed on the 25th day for analysis. (B) Lung tissue was prepared for histological analysis, including morphology and the infiltration of inflammatory cells, by H&E staining. Representative results of H&E staining in the four groups are shown. Scale bar, 200 µm. (C) Lung tissue was prepared for histological analysis. PAS-stained airway cross-sections of each group are shown. Scale, 10 µm. (D) Inflammatory changes were graded by histopathological assessment using a semiquantitative scale of 0–5. The results are presented as the mean ± S.E.M. for each group (n = 4). *p < 0.01 and **p < 0.001 compared with the OVA group. ^###p < 0.001 compared with the mock group.